Huntington's disease (HD) is a devastating neurodegenerative disease caused by expansion of a polyglutamine (polyQ) domain in distinct proteins with different functions. In HD, the polyQ domain is located in the N-terminal region of huntingtin (Htt). This N-terminal region is well conserved in a wide range of species, but polyQ expansion can lead to misfolding and subsequent toxicity of N-terminal fragments of Htt. Since a lack of Htt causes embryonic lethality in mice, Htt is also thought to be essential for animal development and survival. Reducing the expression of mutant Htt is widely accepted as an important strategy for treating HD, so considerable efforts have gone into developing siRNA and anti-sense oligonucleotides to suppress the expression of mutant Htt. These approaches have also raised concerns that markedly suppressing Htt expression could lead to side effects by diminishing the normal function of Htt; however, whether Htt can preserve critical functions without the N-terminal domain that contains the polyQ domain remains unknown. Addressing this issue is important if we are to develop a new strategy to treat HD: if the N-terminal polyQ domain is not required for essential Htt functions and can be removed, complete elimination of the N-terminal region of Htt is now possible since the recent development of the genomic editing tool, CRISPR/Cas9. In this competitive renewal application, we will use CRISPR/Cas9 to investigate the toxicity of N-terminal mutant Htt fragments and therapeutic effects by removing the polyQ-containing N-terminal region. In Aim 1, we will use CRISPR/Cas9 to introduce mutations in the mouse Htt gene in embryos from HD 140Q KI mice to generate truncated mutant Htt genes that express different N-terminal Htt fragments and can be transmitted to offspring via the germline. Using the newly established HD KI mice that express different N-terminal mHtt fragments containing the same polyQ repeat (140Q) at the endogenous level, we will examine the relationship between the length of N-terminal mutant Htt fragments and their nuclear accumulation and toxicity in striatal neurons. In Aim 2, we will use CRISPR/Cas9 to remove the N-terminal polyQ domain as a therapeutic strategy. We will explore whether removing the N-terminal polyQ domain in Htt can eliminate neuropathology without affecting neuronal survival and function in adult mice. These studies will use HD knock-in mice in which mutant Htt is expressed at the same endogenous level as in HD patients. We hope these studies will not only provide new insight into the pathogenesis of N-terminal mutant Htt fragments, but also allow us to develop a novel therapeutic strategy to treat Huntington's disease and other polyQ diseases.